Programmed disassembly of a microtubule-based membrane protrusion network coordinates 3D epithelial morphogenesis in Drosophila

Comprehensive analysis of cellular dynamics during the process of morphogenesis is fundamental to understanding the principles of animal development. Despite recent advancements in light microscopy, how successive cell shape changes lead to complex three-dimensional tissue morphogenesis is still largely unresolved. Using in vivo live imaging of Drosophila wing development, we have studied unique cellular structures comprising a microtubule-based membrane protrusion network. This network, which we name here the Interplanar Amida Network (IPAN), links the two wing epithelium leaflets. Initially, the IPAN sustains cell–cell contacts between the two layers of the wing epithelium through basal protrusions. Subsequent disassembly of the IPAN involves loss of these contacts, with concomitant degeneration of aligned microtubules. These processes are both autonomously and non-autonomously required for mitosis, leading to coordinated tissue proliferation between two wing epithelia. Our findings further reveal that a microtubule organization switch from non-centrosomal to centrosomal microtubule-organizing centers (MTOCs) at the G2/M transition leads to disassembly of non-centrosomal microtubule-derived IPAN protrusions. These findings exemplify how cell shape change-mediated loss of inter-tissue contacts results in 3D tissue morphogenesis.


The EMBO Journal
Ngan Vi Tran et al

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The EMBO Journal © The Author(s) The EMBO Journal Ngan Vi Tran et al

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The EMBO Journal © The Author(s)

Figure
Figure EV2.Time-lapse imaging of cell cycle changes using S/G/M-green in pupal wing epithelial cells between 13 and 16 h APF.(A) Our observations reveal that the majority of cells remain in the S/G2 phase during the early inflation stage (13 h APF at 25 °C).After 3 h (16 h APF at 25 o C), many cells enter mitosis.(B) Delimiting the region of interest (ROI) in which MT protrusion loss and mitoses are counted in dorsal and ventral epithelia.The ROI (magenta box) is adjacent to the trachea (white dashed line) close to the future hinge of the wing.Scale bar: 20 µm (A) and 30 µm (B).

Figure
Figure EV3.Modulating MT stability affects wing morphogenesis.(A) Adult wing in control condition I. (B) Lateral view of αTubulin:GFP of control at 10.5, 12.5, and 14.5 h APF.(C) Adult wing overexpressing hTau in both dorsal and ventral epithelium.(D) Lateral view of αTubulin:GFP during hTau overexpression in both dorsal and ventral layers (nub-Gal4> hTau) at 10.5, 12.5, and 14.5 h APF.(E) Lateral view of αTubulin:GFP during hTau overexpression in dorsal layers only (ap-Gal4> hTau) at 10.5, 12.5, and 14.5 h APF.Note that dorsal protrusions are thicker than ventral protrusions in hTau overexpression only in dorsal cells, which results in loss of cell-cell contacts (arrowheads).(E') Schematics of the loss of cell-cell contacts in hTau oeverexpression only in dorsal cells.Disassembly of MT projections in ventral cells involves degeneration of the basal integrin-laminin complex, but not in dorsal cells, which is sufficient for leading to the loss of cell-cell contact.(F) Adult wing overexpressing Katanin60 in dorsal epithelium.Scale bars: 250 µm (A, C, F), 5 µm (B, D, E).

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Figure EV5.Time lapse images of 3D view of MT protrusions visualized by αTubulin:GFP at 10.5, 12.5, and 14.5 h APF (29 °C) of the wing during conditional knockdown of polo (ap > polo RNAi).(A) Apical surface of the dorsal epithelium is towards the top of the view.(B) Dorsal (top) and ventral (bottom) epithelial cells visualized by αTubulin:GFP (green) and Cnn:RFP (magenta) at 14.5 h APF.Note that mitotic cells are only observed in ventral epithelium (arrowheads).Scale bars: 20 µm (A, B).